This career award will support a period of rigorous training to facilitate the applicant's transition into an independent investigator in academic cardiology. A joint mentorship between Dr. Anthony Rosenzweig, a pioneer in cardiomyocyte physiology, and Dr. Stuart Orkin, a renowned stem cell biologist has been forged to foster the applicant's scientific and career development. To enhance the breadth of the applicant's research training, both didactic and research components will be emphasized. This proposal will address the role of stem and progenitor cells in the biology of early cardiac development and their potential for treating adult myocardial diseases. Recent studies have demonstrated the potential of embryonic and adult stem cells to differentiate into cells of many lineages including beating cardiomyocvtes. The molecular mechanisms involved in cell lineage commitment remains elusive. To gain insights into the earliest events in cardiogenic commitment, a lineage tracing approach was applied to in vitro differentiating embryonic stem (ES) cells to isolate a clonogenic, highly proliferative cell population that is capable of differentiating specifically into beating cardiomyocvtes. Further analysis revealed their expression of stem cell markers such as c-Kit and Sca-1 consistent with their in vitro behavior as cardiogenic stem cells (CSCs). The aims of the proposed research are to 1) Isolate endogenous CSCs from Nkx2.5-EGFP transgenic mice; 2) Characterize the gene expression signature of CSCs during cardiomyogenic differentiation, 3) Identify unique cell surface markers that define CSCs and cardiomyocvtes; and 4) Determine the myocardial regenerative potentials of ES cell-derived CSCs in a myocardial injury-reconstitution model. Completion of these proposed aims will provide an entry into understanding earliest events in cardiac development and evaluate rigorously the potential of stem cell-based myocardial repair. In addition they will help to launch the applicant's career as an independent investigator in the Harvard research community.